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AbstractAged hematopoietic stem cells (HSCs) show reduced reconstitution potential,limiting their use in transplantation settings in the clinic. We demonstrate here that exposure of aged HSCs ex vivo to a pH of 6.9 instead of the commonly used pH of 7.4 results in enhanced HSCs potential that is consistent with rejuvenation,including attenuation of the myeloid bias of aged HSC and restoration of a youthful frequency of epigenetic polarity. Rejuvenation of aged HSCs by pH 6.9 is,at least in part,due to alterations in the polyamine/methionine pathway within pH 6.9 HSCs,and consequently,attenuation of the production of spermidine also attenuated aging of HSCs. Exposure of aged HSCs to pH 6.9,or pharmacological targeting of the polyamine pathway,might thus extend the use of HSCs from aged donors for therapeutic applications. Aged HSCs show compromised reconstitution potential and myeloid bias. We demonstrate that ex‐vivo exposure of aged HSCs to a pH of 6.9 enhances HSC potential and rejuvenation instead of the commonly used pH 7.4. Moreover,pH 6.9 attenuates the myeloid bias of aged HSC via regulating epigenetic polarity to young levels. These effects correspond to a decrease in the polyamine/methionine pathway of spermidine production and via pharmacological inhibitor,DFMO. This study suggests the prospect of using HSCs from aged donors for HSC transplantation therapy. View Publication

The embryonic cell surface is rich in glycosphingolipids (GSLs),which change during differentiation. The reasons for GSL subgroup variation during early embryogenesis remain elusive. By combining genomic approaches,flow cytometry,confocal imaging,and transcriptomic data analysis,we discovered that α1,2-fucosylated GSLs control the differentiation of human pluripotent cells (hPCs) into germ layer tissues. Overexpression of α1,2-fucosylated GSLs disrupts hPC differentiation into mesodermal lineage and reduces differentiation into cardiomyocytes. Conversely,reducing α1,2-fucosylated groups promotes hPC differentiation and mesoderm commitment in response to external signals. We find that bone morphogenetic protein 4 (BMP4),a mesodermal gene inducer,suppresses α1,2-fucosylated GSL expression. Overexpression of α1,2-fucosylated GSLs impairs SMAD activation despite BMP4 presence,suggesting α-fucosyl end groups as BMP pathway regulators. Additionally,the absence of α1,2-fucosylated GSLs in early/late mesoderm and primitive streak stages in mouse embryos aligns with the hPC results. Thus,α1,2-fucosylated GSLs may regulate early cell-fate decisions and embryo development by modulating cell signaling. Synopsis The variation in glycosphingolipids (GSLs) during embryogenesis influences human pluripotent cel differentiation. Specifically,α1,2-fucosylated GSLs control hPC differentiation into germ layers,impacting mesodermal lineage formation through BMP pathway regulation. hESC differentiation towards the three germ layers depends on the levels of α1-2 fucosyltransferase-1 (FUT1).High expression of α1,2-fucosyl GSLs compromises hESC differentiation into mesodermal and cardiac lineages.Overexpression of α1,2-fucosyl GSLs impairs SMAD activation,suggesting they are BMP pathway regulators.FUT1 and FUT2 have different functionalities during early embryonic development. The variation in glycosphingolipids (GSLs) during embryogenesis influences human pluripotent cell differentiation. Specifically,α1,2-fucosylated GSLs control hPC differentiation into germ layers,impacting mesodermal lineage formation through BMP pathway regulation. View Publication

AbstractBruton tyrosine kinase (BTK) is a kinase expressed by various immune cells and is often activated under proinflammatory states. Although the majority of BTK-related research has historically focused on B cells,understanding the role of BTK in non-B cell populations is critical given myeloid cells also express BTK at comparable levels. In this study,we investigated and compared how BTK inhibition in human and murine myeloid cells alters cell phenotype and function. All experiments were performed using two BTK inhibitors (evobrutinib and tolebrutinib) that are currently in late-stage clinical trials for the treatment of multiple sclerosis. Assays were performed to assess the impact of BTK inhibition on cytokine and microRNA expression,phagocytic capacity,and cellular metabolism. In all cells,both evobrutinib and tolebrutinib significantly decreased phosphorylated BTK and LPS-induced cytokine release. BTK inhibition also significantly decreased the oxygen consumption rate and extracellular acidification rate in myeloid cells,and significantly decreased phagocytosis in murine-derived cells,but not human macrophages. To further elucidate the mechanism,we also investigated the expression of microRNAs known to impact the function of myeloid cells. BTK inhibition resulted in an altered microRNA expression profile (i.e.,decreased miR-155-5p and increased miR-223-3p),which is consistent with a decreased proinflammatory myeloid cell phenotype. In summary,these results provide further insights into the mechanism of action of BTK inhibitors in the context of immune-related diseases,while also highlighting important species-specific and cell-specific differences that should be considered when interpreting and comparing results between preclinical and human studies. View Publication

While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known,mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Combined deletion of the histone methyl transferases Prdm3 and Prdm16 in early lung endoderm causes perinatal lethality due to respiratory failure from loss of AT2 cells and the accumulation of partially differentiated AT1 cells. Combination of single-cell RNA-seq,bulk ATAC-seq,and CUT&RUN data demonstrate that PRDM3 and PRDM16 regulate chromatin accessibility at NKX2-1 transcriptional targets critical for perinatal AT2 cell differentiation and surfactant homeostasis. Lineage specific deletion of PRDM3/16 in AT2 cells leads to lineage infidelity,with PRDM3/16 null cells acquiring partial AT1 fate. Together,these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16. Growth and differentiation of pulmonary epithelial cells is precisely controlled to form the alveoli that create the gas exchange region of the lung. Here,the authors demonstrate that epigenetic modulation of the genome by PRDM3/16 mediates NKX2-1 activity to control alveolar cell fate and differentiation during embryonic and perinatal lung development. View Publication

Mucopolysaccharidoses are inherited metabolic disorders caused by the deficiency in lysosomal enzymes required to break down glycosaminoglycans. Accumulation of glycosaminoglycans leads to progressive,systemic degenerative disease. The central nervous system is particularly affected,resulting in developmental delays,neurological regression,and early mortality. Current treatments fail to adequately address neurological defects. Here we explore the potential of human induced pluripotent stem cell (hiPSC)-derived microglia progenitors as a one-time,allogeneic off-the-shelf cell therapy for several mucopolysaccharidoses (MPS). We show that hiPSC-derived microglia progenitors,possessing normal levels of lysosomal enzymes,can deliver functional enzymes into four subtypes of MPS knockout cell lines through mannose-6-phosphate receptor-mediated endocytosis in vitro. Additionally,our findings indicate that a single administration of hiPSC-derived microglia progenitors can reduce toxic glycosaminoglycan accumulation and prevent behavioral deficits in two different animal models of MPS. Durable efficacy is observed for eight months after transplantation. These results suggest a potential avenue for treating MPS with hiPSC-derived microglia progenitors. Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by enzyme deficiencies leading to glycosaminoglycan accumulation and systemic degenerative disease. Here,the authors show that iPSC-derived microglia progenitors can reduce glycosaminoglycan accumulation and prevent behavioral deficits in MPS mouse models. View Publication

Background3D cell culture models have recently garnered increasing attention for replicating organ microarchitecture and eliciting in vivo-like responses,holding significant promise across various biological disciplines. Broadly,3D cell culture encompasses organoids as well as single- and multicellular spheroids. While the latter have found successful applications in tumor research,there is a notable scarcity of standardized intestinal models for infection biology that mimic the microarchitecture of the intestine. Hence,this study aimed to develop structured multicellular intestinal spheroids (SMIS) specifically tailored for studying molecular basis of infection by intestinal pathogens.ResultsWe have successfully engineered human SMIS comprising four relevant cell types,featuring a fibroblast core enveloped by an outer monolayer of enterocytes and goblet cells along with monocytic cells. These SMIS effectively emulate the in vivo architecture of the intestinal mucosal surface and manifest differentiated morphological characteristics,including the presence of microvilli,within a mere two days of culture. Through analysis of various differentiation factors,we have illustrated that these spheroids attain heightened levels of differentiation compared to 2D monolayers. Moreover,SMIS serve as an optimized intestinal infection model,surpassing the capabilities of traditional 2D cultures,and exhibit a regulatory pattern of immunological markers similar to in vivo infections after Campylobacter jejuni infection. Notably,our protocol extends beyond human spheroids,demonstrating adaptability to other species such as mice and pigs.ConclusionBased on the rapid attainment of enhanced differentiation states,coupled with the emergence of functional brush border features,increased cellular complexity,and replication of the intestinal mucosal microarchitecture,which allows for exposure studies via the medium,we are confident that our innovative SMIS model surpasses conventional cell culture methods as a superior model. Moreover,it offers advantages over stem cell-derived organoids due to scalability and standardization capabilities of the protocol. By showcasing differentiated morphological attributes,our model provides an optimal platform for diverse applications. Furthermore,the investigated differences of several immunological factors compared to monotypic monolayers after Campylobacter jejuni infection underline the refinement of our spheroid model,which closely mimics important features of in vivo infections.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13099-024-00644-6. View Publication

Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. Although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals,the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence,we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions (DMRs) when compared to memory CD8+ T cells. Among the top demethylated DMRs in cytomegalovirus-specific CD8+ T cells was TBKBP1,coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells,we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with higher TBKBP1 expression at both mRNA and protein level,independent of alternative splicing of TBKBP1 transcripts. Notably,the distinct DNA methylation patterns in CD8+ T cell subsets was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively,our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies. Author summaryHuman cytomegalovirus (CMV) is a herpesvirus that infects a significant portion of the global population. While it usually causes asymptomatic or mild infections,CMV can have severe consequences for individuals with a weakened immune system,such as stem cell or organ transplant recipients or individuals with HIV/AIDS. The immune response to CMV is characterized by expansion of virus-specific CD8+ T cells,which recognize and eliminate CMV-infected cells,thereby controlling the infection. Studies have shown that the pathogen-induced differentiation of naive to effector memory CD8+ T cells is accompanied by epigenetic changes. In order to identify the major genes involved in the functionality of CMV-specific CD8+ T cells,which are also regulated by DNA methylation,we compared the methylation profiles of CMV-specific CD8+ T cells with memory CD8+ T cells. As a result of this,we found that TBK-binding protein 1 (TBKBP1) plays a crucial role in the function of CMV-specific CD8+ T cells and enhances virus neutralization capacity upon overexpression,which has not been reported previously. This study opens the possibility of not only identifying unknown genes that contribute to the functionality of CMV-specific CD8+ T cells,but also potentially lead to improvements in adoptive T cell therapies. View Publication

CD38 has emerged as a potential therapeutic target for patients with systemic lupus erythematosus (SLE) but it is not known whether CD38 alters CD4+ T cell function. Using primary human T cells and CD38-sufficient and CD38-deficient Jurkat T cells,we demonstrate that CD38 shifts the T cell lipid profile of gangliosides from GM3 to GM2 by upregulating B4GALNT1 in a Sirtuin 1-dependent manner. Enhanced expression of GM2 causes ER stress by enhancing Ca2+ flux through the PLCγ1-IP3 pathway. Interestingly,correction of the calcium overload by an IP3 receptor inhibitor,but not by a store-operated calcium entry (SOCE) inhibitor,improves IL-2 production by CD4+ T cells in SLE. This study demonstrates that CD38 affects calcium homeostasis in CD4+ T cells by controlling cell membrane lipid composition that results in suppressed IL-2 production. CD38 inhibition with biologics or small drugs should be expected to benefit patients with SLE. CD38 has been reported to be upregulated on T cells in lupus with unclear functional consequences. Here the authors show that CD4+CD38+ T cells from lupus patients have altered calcium homeostasis accompanied by alteration in cell membrane lipid composition and a suppression of IL-2. View Publication

Our examination of RNA helicases for effects on HIV-1 protein production and particle assembly identified Rocaglamide (RocA),a known modulator of eIF4A1 function,as an inhibitor of HIV-1 replication in primary CD4+ T cells and three cell systems. HIV-1 attenuation by low-nM RocA doses was associated with reduced viral particle formation without a marked decrease in Gag production. Rather,the co-localization of Gag and HIV-1 genomic RNA (gRNA) assemblies was impaired by RocA treatment in a reversible fashion. Ribonucleoprotein (RNP) immunoprecipitation studies recapitulated the loss of Gag-gRNA assemblies upon RocA treatment. Parallel biophysical studies determined that neither RocA nor eIF4A1 independently affected the ability of Gag to interact with viral RNA,but together,they distorted the structure of the HIV-1 RNP visualized by electron microscopy. Taken together,several lines of evidence indicate that RocA induces stable binding of eIF4A1 onto the viral RNA genome in a manner that interferes with the ordered assembly of Gag along Gag-gRNA assemblies required to generate infectious virions. View Publication

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically,dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs,followed by either hyperoxic (20 % O2) or physioxic (5 % O2) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism),higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity,higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly,release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint,amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups,physioxic hiPSCs revealed lower polarized mitochondrial membrane potential,altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V,respectively. Taken together,physioxic culture of hiPSCs improved chromosomal stability,which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition. Graphical abstractImage 1 View Publication

IntroductionEndothelial progenitor cells (EPCs) are essential for maintenance of vascular homeostasis and stability,key processes in the pathogenesis of systemic lupus erythematosus (SLE). However,the role and phenotypic characterization of EPCs populations in SLE have not been completely elucidated.ObjectiveTo identify EPCs specific subpopulations in patients with SLE using a novel flow cytometry tool.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from patients with SLE and healthy controls (HC). mRNA and surface protein expression were determined by quantitative PCR (qPCR) and flow cytometry. Clusters identification and characterization were performed using tSNE-CUDA dimensionality reduction algorithms.ResultstSNE-CUDA analysis identified eight different clusters in PBMCs from HC and patients with SLE. Three of these clusters had EPC-like phenotype and the expression was elevated in patients with SLE. Moreover,four SLE-associated subclusters were found mainly expressed in patients with SLE,being only present in patients in remission with SLE and significantly associated with the 2021 Definition of Remission in SLE. Importantly,we also identified specific clusters in SLE patients with organ damage,according to the Systemic Lupus International Collaborating Clinics (SLICC)/American College of Rheumatology damage index (SDI). These clusters showed an EPC-like phenotype,but the expression of angiogenic markers was lower compared to HC or patients without organ damage,suggesting an impaired angiogenic function.ConclusionOur novel approach identified clusters of EPCs in patients with SLE that are associated with remission and damage. Therefore,these clusters might be useful biomarkers to predict disease progression and severity in SLE pathogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13075-024-03397-4. Key messages Novel endothelial progenitor cells clusters have been characterized in patients with SLE.Endothelial progenitor cells clusters are potential biomarkers of damage and remission in SLE. Supplementary InformationThe online version contains supplementary material available at 10.1186/s13075-024-03397-4. View Publication

BackgroundAlthough several cell culture systems have been developed to investigate the function of the mammary gland in dairy livestock,they have potential limitations,such as the loss of alveolar structure or genetic and phenotypic differences from their native counterparts. Overcoming these challenges is crucial for lactation research. Development of protocols to establish lactating organoid of livestock represents a promising goal for the future. In this study,we developed a protocol to establish a culture system for mammary organoids in dairy goats to model the mammary gland development and lactation process.ResultsThe organoids cultured within an extracellular matrix gel maintained a bilayer structure that closely resembled the native architecture of mammary tissue. The expansion of mammary organoids was significantly promoted by growth factors containing epidermal growth factor and fibroblast growth factor 2 whereas the proliferative index of the organoids was significantly inhibited by the treatment with WNT inhibitors. Upon stimulation with a lactogenic medium containing prolactin,the mammary organoids exhibited efficient lactation,characterized by the accumulation of lipid droplets in the lumen space. The lactation could be sustained for more than 3 weeks. Importantly,the expression patterns of genes related to fatty acid synthesis and milk proteins in lactating organoids closely mirrored those observed in mammary tissues. These observations were confirmed by data from proteomic analysis that the bulk of milk proteins was produced in the lactating organoids.ConclusionThis study is the first to establish a mammary organoid culture system modeling the mammary gland development and lactation process in ruminants. The efficient induction of lactation in ruminant mammary organoids holds promises for advancing the field of cell-based milk bio-manufacture in the food industry.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40104-024-01084-7. View Publication